human identification applications on ion torrent pgm™
DESCRIPTION
Presented at South Africa User Meeting in December 2012 by Lisa Calandro Director, Product Management Human Identification (Life Technologies)TRANSCRIPT
1/10/2013 | © Life Technologies 1
Human Identification Applications on Ion Torrent PGM™
Lisa Calandro Director, Product Management Human Identification, Life Technologies Special Acknowledgments Dr. Walther Parson, Institute of Legal Medicine, University of Innsbruck, Austria Dr. Kenneth Kidd, Department of Genetics, Yale University School of Medicine, USA
1/10/2013 | © Life Technologies 2
An Overview of PGM™ Technology
Development of the PGM™ HID SNP Panel
Mitochondrial Sequencing on the PGM™
Future Development Opportunities
Agenda
09/23/12
An Overview of PGM™ Technology
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Sequencing Instrument
Ion OneTouch™ Instruments Emulsion PCR and Enrichment
Semiconductor Chip
Sequencing Chemistry Natural nucleotides Natural enzymes
Sample Prep Libraries Clonal beads
Torrent Server
The Ion Torrent PGM™ Instrument System
INSTRUMENTS REAGENTS DATA ANALYSIS
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Sequence Detection by pH
1/10/2013 | © Life Technologies 6
Workflow and Technology Summary
Confidential and Proprietary—DO NOT DUPLICATE 6
1/10/2013 | © Life Technologies 7
Ion AmpliSeq™ Technology: As Simple As PCR Ultra-high multiplex PCR for targeted resequencing
Up to 3, 072 primer pairs per tube
Starting from just 10ng DNA
From DNA to annotated variants in as little as 10 hours
7
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8
Ion AmpliSeq™ Technology How does it work?
Up to 3000 primers
10ng/rxn or X ng for x pools Ultra-Multiplex
PCR
Amplification
Phosphorylation
Adaptaters ligation Template Sequence Analysis Library
1/10/2013 | © Life Technologies 10
Demonstration of the Chemistry
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Sequencing: Flows and Cycles
Confidential and Proprietary -- Do Not Duplicate
• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step • A “cycle” is four consecutive dNTP flows: T-A-C-G = 1 cycle
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
Sequence of Interest Key Sequence
T A C G T A C G T A C G T A C G T A C G
Cycle 1 Cycle 3 Cycle 2 … etc
T
A
C
G
Cycle 1
...---Ion Sphere™
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T
Sequencing: Flows and Cycles
Confidential and Proprietary -- Do Not Duplicate
• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step • A “cycle” is four consecutive dNTP flows: T-A-C-G = 1 cycle
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
Sequence of Interest Key Sequence
C
T A C G T A C G T A C G T A C G T A C G
Cycle 1 Cycle 3 Cycle 2 … etc
T
A
C
G
Cycle 2
...---Ion Sphere™
1/10/2013 | © Life Technologies 13
T
Sequencing: Flows and Cycles
Confidential and Proprietary -- Do Not Duplicate
• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step • A “cycle” is four consecutive dNTP flows: T-A-C-G = 1 cycle
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
Sequence of Interest Key Sequence
C
T A C G T A C G T A C G T A C G T A C G
Cycle 1 Cycle 3 Cycle 2 … etc
T
A
C
G
T
Cycle 3+
A G ...---Ion Sphere™
1/10/2013 | © Life Technologies 14
T
Sequencing: Flows and Cycles
Confidential and Proprietary -- Do Not Duplicate
• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step • A “cycle” is four consecutive dNTP flows: T-A-C-G = 1 cycle
Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G
Sequence of Interest Key Sequence
A C G
T A C G T A C G T A C G T A C G T A C G
Cycle 1 Cycle 3 Cycle 2 … etc
T
A
C
G
T
T T C G C A G G G T A C
And so on…
...---Ion Sphere™
1/10/2013 | © Life Technologies 15
Must be read “up-and-down” along with “left-to-right”
Height of bar indicates how many nucleotides incorporated during flow
Data Output: Ionogram
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Amplicon-based sequencing more sensitive than other assay systems
Amenable to degraded DNA (down to 75 bp)
Multiplex individuals – Barcodes (up to 96)
Faster library construction
Automated enrichment system
Very fast sequencing time
Less total hands-on time
Higher throughput (runs per week)
Natural chemistry (less bias, less maintenance) – no laser or camera
Why PGM™ Sequencing?
09/23/12
Development of the PGM™ HID SNP Panel
1/10/2013 | © Life Technologies 18
SNP Classes
Identity SNPs Lineage-
informative SNPs
Ancestry-informative
SNPs
High Heterozygosity Low Population Heterogeneity
Phenotypic SNPs
Haplotype Markers for Kinship Analysis
High Population Heterogeneity
Hair, Eye, Skin Colour
Budowle BioT 2008
RUO v0.1 released 7/2012
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Based on published SNPs with high heterozygosity and low Fst
Genotype match probabilities of 10-31 - 10-35
HID SNP Panel v0.1 (Identity Panel)
136 SNP panel
33 Y - SNP
70 - Ken Kidd
36 - SNPforID
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Library Preparation
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Data Analysis
Torrent Server Torrent Browser Run Analysis Application-Specific Plugins
Third-Party Desktop Tools
1/10/2013 | © Life Technologies 22
Panel Development Tools: Amplicon Coverage
Total Number of Amplicon Reads (Sorted) [Grey bar denoted 1 SD from the mean (red line)]
Sorted Amplicon
Num
ber o
f Rea
ds
Total Number of Amplicon Reads (Sorted) [Grey bar denoted 1 SD from the mean (red line)]
Sorted Amplicon
Num
ber o
f Rea
ds
Female Individual Male Individual
Y SNPs Y SNPs
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SNP Genotype Call Viewer: Integrative Genomics Viewer (IGV)
rs891700
Coverage
Alignments
A
Het G/A
Reference
T C G
1/10/2013 | © Life Technologies 24
HID-SNP Genotyper Plugin
1/10/2013 | © Life Technologies 25
Allele Coverage
Alleles Represented
SNPs by rs ID
HID-SNP Genotyper Plugin
1/10/2013 | © Life Technologies 27
Next-generation sequencing technologies provide allow high levels of multiplexing for both SNPs and individuals
Development of the HID SNP Panel v0.1, an Identity SNP panel on the PGM™ sequencer using well-characterized polymorphisms
HID SNP Panel v0.1 demonstrates 99% concordance with Ken Kidd controls
HID-SNP Genotyper Plugin utility built to assist display and data evaluation
Summary
1/10/2013 | © Life Technologies 33
Sunshine
Ion Community Web Page
ioncommunity.lifetechnologies.com\
1/10/2013 | © Life Technologies 34
Continue external collaborations to research potential applications on the PGM™ sequencer
Develop ancestral/phenotypic and lineage SNP panels
Develop small amplicon mitochondrial control region assay
Investigate applications within Microbial Forensics
Future Plans
1/10/2013 | © Life Technologies 35
Acknowledgments
Robert Lagacé Sharon Wootton Reina Langit Joe Chang Chien-Wei Chang Narasimhan Rajagopalan
1/10/2013 | © Life Technologies 36 09/23/12
Thank You
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